Antibodies specific for human thymosin β15 protein and uses thereof

ABSTRACT

The present inventors have now discovered that humans have a gene that encodes a novel protein of the thymosin β family. This novel protein, herein referred to as thymosin β15, has the ability to bind and sequester G-actin, like other members of the thymosin β family, but unlike what is known about other members it also directly regulates cell motility in prostatic carcinoma cells. The present invention is direct to an isolated cDNA encoding the human thymosin β15 gene (SEQ ID NO: 1) and have deduced the amino acid sequence (SEQ ID NO: 2).

This application is a divisional of application Ser. No. 09/069,484filed on Apr. 29, 1998, now U.S. Pat. No. 6,017,717 which is adivisional of application Ser. No. 08/931,877 filed on Sep. 17, 1997 nowU.S. Pat. No. 5,831,033, granted Nov. 3, 1998, which is a divisional ofapplication Ser. No. 08/801,796 filed on Feb. 14, 1997, now U.S. Pat.No. 5,721,337, granted Feb. 24, 1998, which is a divisional ofapplication Ser. No. 08/664,856 filed on Jun. 17, 1996, now U.S. Pat.No. 5,663,071, granted Sep. 2, 1997.

HUMAN THYMOSIN β15 GENE, PROTEIN AND USES THEREOF

The work described herein was supported, in part, by National Institutesof Health grant CA37393. The U.S. Government has certain rights to thisinvention.

BACKGROUND OF THE INVENTION

The present invention provides novel genes, proteins, and uses thereofincluding, methods for diagnosing and treating cancer, particularlymetastatic cancer.

Most eukaryotic cells (execptions include red blood cells and adultmuscles) contain high concentrations, i.e., up to ˜250 μmol/l, ofmomomeric actin. How such actin remains unpolymerized in the cytoplasmhas remained a problem in cell biology (Nachmiar, V., Current Opinion inCell Biology, 1993, 5:56). Profilin, originally thought to be theactin-sequestering protein, is not present in sufficient amounts toaccount for more than part of the monomeric actin levels observed.Recently, an actin-sequestering 5 kD peptide was discovered in highconcentration in human platelets (Safer, et al., Proc. Natl. Acad. SciUSA 1990 87:2536-2540) and shown to be identical to a previously knownpeptide (Safter, et al., J. BIol. Chem., 1991, 268:4029-4032) originallythought to be the thymic hormone, thymosin β₄ (Tβ₄) (D. Safer, J. MuscleRes. Cell Motil, 1992.13:269-271). A detailed kinetic study of theinteraction of Tβ₄ and actin (Weber, et al., Biochemistry 1992,31:6179-6185)), together with other studies (Yu, et al., J. BIol. Chem.,1993, 268:502-509 and Cassimelds, et al., J. Cell Biol., 1992,119:1261-1270) support the hypothesis that Tβ₄ and Tβ₁₀ function primaryas G-actin buffers. Unpublished data (E. Hannappel) extend the functionto several other, β thymosins. Tβ₄ has also been shown to inhibitnucleotide exchange by actin, whereas profilin increases the rate ofexchange (Coldschmidt-Clermont, et al., Mol. Cell Bio., 1992,3:1015-1025).

All vertebrates studied contain one or often two, β-thymosins. Thus, themembers of the β-thymosin family are believed to be important in allspecies. Three new family members (Low, et al., Arch. Biochem. Biophys.,1992, 293:32-39 and Schmid, B., Ph.D Thesis, University of Tubingen1989) have been found in perch, trout and in sea urchin, the firstnon-vertebrate source. The sequences are well conserved suggesting thatactin sequestration is probably a property of all β-thymosins. However,when Tβ₄ was discovered and its sequence first determined in 1981 (Low,et al., Proc. Natl. Acad. Sci., USA 1981, 78:1162-1166), data werepresented that suggested two extracellular functions (Low, et al. supraand Rebar, et al., Science 1981, 214:669-671). Two recent papersindicate a different and unexpected effect of a tetrapeptide which maybe derived from the amino terminus of Tβ₄.

Several reports demonstrate regulation of Tβ₄ or Tβ₁₀ synthesis at thetranscriptional or translational level. An interferon-inducible gene(Cassimelds, et al., J. Cell. Biol. 1992, 119:1261-1270 and Sanders, etal., Proc. Natl. Acad. Sci. USA 1992, 89:4678-4682) is identical to thecDNA of human Tβ₄, and there are several genes for Tβ₄ in humans.(Clauss, et al., Genomies 1991, 9:75-180 and Gomez-Marquez, et al., J.Immunol. 1989, 143:2740-2744)

It would be desirable to identify new members of the β-thymosin family,particularly in humans.

Bao and Zetter reported in an abstract presented at the AmericanAssociation for Cancer Research annual meeting (Mar. 18-22, 1995) thedifferential expression of a novel mRNA expressed in high-metastatic rattumor cell lines, but not in a low metastatic variant. cDNA was isolatedand was reported to encode a protein with 68% identity to the ratthymosin,64. However, the nucleotide sequence and the deduced amino acidsequence were not reported.

SUMMARY OF THE INVENTION

We have now discovered that humans have a gene that encodes a novelprotein of the thymosin β family. This novel protein, herein referred toas thymosin β15, has the ability to bind and sequester G-actin, likeother members of the thymosin β family, but unlike what is known aboutother members it also directly regulates cell motility in prostaticcarcinoma cells. We have isolated a cDNA of the human thymosin β15 gene(SEQ ID NO: 1) and have deduced the amino acid sequence (SEQ ID NO: 2).We have shown that enhanced transcripts (mRNA) and expression of thethymosin β15 gene in non-testicular cells has a high correlation todisease state in a number of cancers, such as prostate, lung, melanomaand breast cancer, particularly metastatic cancers. Accordingly,discovering enhanced levels of transcript or gene product innon-testicular tissues can be used in not only a diagnostic manner, buta prognostic manner for particular cancers.

The present invention provides isolated nucleic acids (polynucleotides)which encode thymosin β15 having the deduced amino acid sequence of SEQID. NO: 2 or a unique fragment thereof. The term “unique fragment”refers to a portion of the nucleotide sequence or polypeptide of theinvention that will contain sequences (either nucleotides or amino acidresidues) present in thymosin β15 (SEC ID NO: 2) but not in other memberof the thymosin family. This can be determined when the hybridizationprofile of that fragment under stringent conditions is such that it doesnot hybridize to other members of the thymosin family. Such fragmentscan be ascertained from FIG. 3. A preferred set of unique fragments arethose that contain, or contain polynucleotides that encode, amino acid 7to 12 of SEQ ID NO: 2, amino acid 21 to 24 of SEQ ID NO: 2 and aminoacid 36 to 45 of SEQ ID NO: 2. Preferably, the unique nucleotidesequence fragment is 10 to 60 nucleotides in length, more preferably, 20to 50 nucleotides, most preferably, 30 to 50 nuceotides. Preferably, theunique polypeptide sequence fragment is 4 to 20 amino acids in length,more preferably, 6 to 15 amino acids, most preferably, 6 to 10 aminoacids.

The polynucleotides of the present invention may be in the form of RNAor in the form of DNA, which DNA includes cDNA, genomic DNA, andsynthetic DNA. The DNA may be double-stranded or single-stranded, and ifsingle stranded may be the coding strand or non-coding (anti-sense)strand. The coding sequence which encodes the mature polypeptides may beidentical to the coding sequence shown in SEQ ID NO: 1 or may be adifferent coding sequence which coding sequence, as a result of theredundancy or degeneracy of the genetic code, encodes the same proteinas the DNA of SEQ ID NO: 1.

The polynucleotide may have a coding sequence which is a naturallyoccurring allelic variant of the coding sequence shown in SEQ ID NO: 1.As known in the art, an allelic variant is an alternate form of apolynucleotide sequence which may have a substitution, deletion oraddition of one or more nucleotides, which does not substantially alterthe function of the encoded protein.

The present invention also provides an isolated polynucleotide segmentwhich hybridize under stringent conditions to a unique portion of thehereinabove-described polynucleotides, particularly SEQ ID NO:1. Thesegment preferably comprises at least 10 nucleotides. As herein used,the term “stringent conditions” means hybridization will occur only ifthere is at least 95% and preferably at least 97% identity between thesequences. These isolated segments may be used in nucleic acidamplification techniques, e.g., PCR, to identify and/or isolatepolynucleotides encoding thymosin β15.

As used herein a polynucleotide “substantially identical” to SEQ ID NO:1is one comprising at least 90% homology, preferably at least 95%homology, most preferably 99% homology to SEQ ID NO: 1. The reason forthis is that such a sequence can encode thyfnosin β15 in multiplemammalian species.

The present invention further provides an isolated and purified humanthymosin β15 having the amino acid sequence of SEC ID NO: 2, or a uniquefragment thereof, as well as polypeptides comprising such uniquefragments, including, for example, amino acid 7 to 12 of SEQ ID NO: 2,amino acid 21 to 24 of SEQ ID NO: 2 and amino acid 36 to 45 of SEQ IDNO: 2.

In accordance with yet another aspect of the present invention, thereare provided isolated antibodies or antibody fragments which selectivelybinds human thymosin β15. The antibody fragments include, for example,Fab, Fab′, F(ab′)2 or Fv fragments. The antibody may be a single chainantibody, a humanized antibody or a chimeric antibody.

The term “isolated” means that the material is removed from its originalenvironment (e.g., the natural environment if it is naturallyoccurring). For example, a naturally-occurring polynucleotides orpolypeptides present in a living animal is not isolated, but the samepolynucleotides or DNA or polypeptides, separated from some or all ofthe coexisting materials in the natural system, is isolated. Suchpolynucleotides could be part of a vector and/or such polynucleotides orpolypeptides could be part of a composition, and still be isolated inthat such vector or composition is not part of its natural environment.

The present invention also relates to vectors which includepolynucleotides of the present invention, host cells which aregenetically engineered with vectors of the invention and the productionof polypeptides of the invention by recombinant techniques.

The present invention further provides a method of treating a neoplasticcell expressing human thymosin, β15 by administering to the cell aneffective amount of a compound which suppresses the activity orproduction of the human thymosin β15. Preferably, the compoundinterferes with the expression of the human thymosin β15 gene. Suchcompounds include, for example, antisense oligonucleotides, ribozymes,antibodies, including single chain antibodies and fragments thereof.

DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B show differential mRNA display and Northern analysis ofDunning R-3327 rat prostatic adenocarcinoma variants. Total RNA fromAT2.1 (lane 1), AT3.1 (lane 2) and AT6.1 (lane 3) cells were reverse-transcribed and amplified by PCR with a primer set, T₁₁ AG and a 10 merAGGGAACGAG (SEQ ID NO:3) in the presence of [α35-S]dATP. The PCRfragments were displayed on a 6% polyacrylamide gel andautoradiographed. The differentially expressed band is indicated byarrowhead. B. Northern blot analysis of thymosin β15 gene. Two μg ofpoly (A) RNA was isolated from Dunning R-3327 variants AT2. l (lane 1),AT3.1 (lane 2), AT6.1 (lane 3), and Mat Lylu (lane 4), fractionated on a1.1% formaldehyde-agarose gel, transferred to Hybond-N+ nylon membrane(Amersham) and hybridized with a random primed (Grillon C, et al., FEBS1990, 274:30-34) ³²P-labeled Tβ15 Tβ15 cDNA fragment. The same blot washybridized with a rat β-actin probe to demonstrate that equivalentamounts of RNA were loaded in each lane.

FIG. 2 is the nucleotide sequence (SEQ ID NO.: 1) of Tβ15 cDNA and thepredicted amino acid sequence (SEQ ID NO.: 2) (single-letter code). Thesequence numbers of the nucleotides and amino acids are indicated on theright side of the sequences. The translation initiation codon ATG isunderlined, and the termination codon TAA is marked with an asterisk. Aputative actin binding region is underlined. These sequence data areavailable from GenBank under accession number U25684.

FIG. 3 shows the alignment of the deduced Tβ15 protein sequence and someof the other β thymosin isoforms. Regions of amino acid identity arerepresented by white letters boxed in black. Unboxed black letterscorrespond to nonidentical regions. Dots correspond to gaps introducedin the sequence to optimize alignment.

FIG. 4 shows expression of Tβ15 in various rat tissues. Themultiple-tissue blot was obtained from Clontech. The blot was hybridizedwith the Tβ15 cDNA probe. Rat GAPDH is a loading control.

FIGS. 5A and 5B show in situ hybridization with antisense riboprobe forTβ15 on prostatic adenocarcinoma patients. FIG. 5A shows differentialexpression in tumors. The small arrow shows positive staining. The largearrow shows negative staining. FIG. 5B shows that in poorlydifferentiated and invasive prostate carcinoma, single cells invadingstroma display intense staining (arrow).

FIGS. 6A, 6B and 6C show the effect of Tβ15 on actin polymerization.

FIG. 6A. 3 μM of pyrene-labeled G-actin was polymerized in the presenceof various amounts of GST-Tβ4 fusion peptide (▾), GST-Tβ15 (▴) or GSTalone (∘). The final extent of polymerization was determined from thefinal levels of pyrene-labeled actin (fluorescence). All solutionscontained 5.5 mM Tris, pH7.6, 167 μM CaCl₂, 0.5 mM glutathione, 167 μMDTT, and 420 μM ATP. Polymerization was induced by addition of 2 mMMgCl₂ and 150 mM KCl. Error bars denote the range of duplicatemeasurements made from separate dilutions of the fusion proteins.

FIG. 6B. 2 μM of pyrene-labeled G-actin was polymerized in the presenceof various amounts of monomeric Tβ15 that had been cleaved from GST bythrombin. The relative rates of polymerization were derived from themaximal rate of fluorescence increase in the initial phase ofpolymerization.

FIG. 6C. The final extent of actin assembly was determined by the samemethods used for the thymosin GST fusion peptides. Experimentalconditions are those described for FIG. 6B.

FIGS. 7A, 7B and 7C show serum stimulated migration of controltransfected and Tβ15 transfected Dunning R-3327 variants and theirgrowth rate. FIG. 7A. Vector control transfected (∘, ∇) and Tβ15antisense (, ▾) transfected AT3.1 cell clones. FIG. 7B. Vector controltransfected (∘, ∇) and Tβ15 sense transfected (, ▾) AT2.1 cell clones.Data are expressed as the mean ±SE (n=4). FIG. 7C. Growth curves ofcontrol transfected and Tβ15 (sense or antisense) transfected DunningR-3327 clones. Cells from vector control transfected AT2.1 (∘), Tβ15sense transfected AT2.1 (), vector control transfected AT3.1 (∇) andTβ15 antisense transfected AT3.1 (▾) were plated at initial 10⁴cells/well in RPMl 1640 with 10% FBS and 250 nM dexamethasome in 12-wellplates. Cells were harvested and counted at indicated times. Pointsrepresent the mean ±SE (n=3).

FIGS. 8A and 8B show Western analysis of thymosin β-GST fusion protein.FIG. 8A is a Coomasie staining of GST-Tβ fusion proteins. FIG. 8B is aWestern analysis of GST-Tβ fusion proteins with affinity purifiedanti-Tβ15 C-terminal peptide antibody. Lane 1: GST-Tβ4; Lane 2:GST-Tβ15; Lane 3: GST only

FIG. 9 shows a Northern analysis of thymosin β15 in mouse lung tumorcells. LA-4: mouse lung adenoma cell line; M27 and H59: metastaticvariants derived from mouse Lewis lung adenocarcinoma cell line.Northern blot analysis revealed that the probe detected the thymosin β15mRNA expression in M27 cells, less expression in H59 cells, but noexpression in LA-4 cells.

FIG. 10A, 10B, 10C and 10D show immunohistochemical staining of humanprostatic carcinoma tissues with an affinity purified polyclonalantibody to thymosin β15. A. Nonmalignant prostatic epithelia (largearrow) and high-grade prostatic intraepithelial neoplasia (PIN) (smallarrow). B. Moderately differentiated prostatic carcinoma showingheterogeneoue immunostaining (small arrow, positive; large arrow,negative). C. Poorly differentiated prostatic carcinoma. D. Single cellsinvading stroma showing intense staining.

FIG. 11 is a 1.4% agarose gel electrophoresis of RT-PCR amplified βthymosins from the rat prostatic cell lines. Lane1, weakly metastaticAT2.1; lane 2, 3 and 4, highly metastatic AT3.1, AT6.1 and Mat Lylu;lane 5 and 6, nonmetastatic NbE and MC2; lane 7, weakly metastatic Fb2.β-actin PCR was used as internal control of each sample.

DETAILED DESCRIPTION OF THE INVENTION

A well characterized series of cell lines that show varying metastaticpotential has been developed from the Dunning rat prostatic carcinoma(Isaacs, et al., Prostate 9, 261-281 and Bussebakers, et al., CancerRes. 52,2916-2922 (1992)). Coffey and colleagues previously showed adirect correlation between cell motility and metastatic potential in theDunning cell lines (Mohler, et al., Cancer Res. 48, 4312-4317 (1988),Parin, et al., Proc. Natl. Acad. Sci, USA 86, 1254-1258 (1989) andMohler, et al., Cancer Metast. Rev 12, 53-67 (1993)). We compared geneexpression in poorly metastatic and highly metastatic cell lines derivedfrom Dunning rat prostate carcinoma using differential mRNA display. Theresults of these studies revealed the expression of a novel member ofthe thymosin beta family of actin-binding molecules, thymosin β15. Usingthis information, we isolated and sequenced a cDNA encoding humanthymosin β15.

Although members of the thymosin β family have been shown to bind andsequester G-actin, they have not previously been demonstrated to altercell motility. Our studies, however, reveal that this new member,thymosin β15, directly regulates cell motility in prostatic carcinomacells. We have shown that expression of thymosin β15 is upregulated inhighly metastatic prostate cancer cell lines relative to poorlymetastatic or nonmetastatic lines. In addition, thymosin β15 wasexpressed in human prostate carcinoma specimens but not in normal humanprostate. Although not wishing to be bound by theory, this indicatesthat β15 plays a role in the process of metastatic transformation.

The present invention provides a polynucleotide sequence encoding all orpart of thymosin β15 having the deduced amino acid sequence of SEQ IDNO:2 or a unique fragment thereof. A nucleotide sequence encoding humanthymosin β15 is set forth as SEQ ID NO:1.

The sequences of the invention may also be engineered to providerestriction sites, if desired. This can be done so as not to interferewith the peptide sequence of the encoded thymosin β15, or may interfereto any extent desired or necessary, provided that the final product hasthe properties desired.

Where it is desired to express thymosin β15 or a unique fragmentthereof, any suitable system can be used. The general nature of suitablevectors, expression vectors and constructions therefor will be apparentto those skilled in the art.

Suitable expression vectors may be based on phages or plasmids, both ofwhich are generally host-specific, although these can often beengineered for other hosts. Other suitable vectors include cosmids andretroviruses, and any other vehicles, which may or may not be specificfor a given system. Control sequences, such as recognition, promoter,operator, inducer, terminator and other sequences essential and/oruseful in the regulation of expression, will be readily apparent tothose skilled in the art, and may be associated with the naturalthymosin β15 or with the vector used, or may be derived from any othersource as suitable. The vectors may be modified or engineered in anysuitable manner.

Correct preparation of nucleotide sequences may be confirmed, forexample, by the method of Sanger et al. (Proc. Natl. Acad. Sci. USA74:5463-7 (1977)).

A cDNA fragment encoding the thymosin β15 of the invention may readilybe inserted into a suitable vector. Ideally, the receiving vector hassuitable restriction sites for ease of insertion, but blunt-endligation, for example, may also be used, although this may lead touncertainty over reading frame and direction of insertion. In such aninstance, it is a matter of course to test transformants for expression,1 in 6 of which should have the correct reading frame. Suitable vectorsmay be selected as a matter of course by those skilled in the artaccording to the expression system desired.

By transforming a suitable organism or, preferably, eukaryotic cellline, such as HeLa, with the plasmid obtained, selecting thetransformant with ampicillin or by other suitable means if required, andadding tryptophan or other suitable promoter-inducer (such asindoleacrylic acid) if necessary, the desired thymosin β15 may beexpressed. The extent of expression may be analyzed by SDSpolyacrylamide gel electrophoresis-SDS-PAGE (Lemelli, Nature 227:680-685(1970)).

Suitable methods for growing and transforming cultures etc. are usefullyillustrated in, for example, Maniatis (Molecular Cloning, A LaboratoryNotebook, Maniatis et al. (eds.), Cold Spring Harbor Labs, N.Y. (1989)).

Cultures useful for production of thymosin β15, or a peptide thereof,may suitably be cultures of any living cells, and may vary fromprokaryotic expression systems up to eukaryotic expression systems. Onepreferred prokaryotic system is that of E. coll, owinfg to its ease ofmanipulation. However, it is also possible to use a higher system, suchas a mammalian cell line, for expression of a eukaryotic protein.Currently preferred cell lines for transient expression are the HeLa andCos cell lines. Other expression systems include the Chinese HamsterOvary (CHO) cell line and the baculovirus system.

Other expression systems which may be employed include streptomycetes,for example, and yeasts, such as Saccharomyces spp., especially S.cerevisiae. Any system may be used as desired, generally depending onwhat is required by the operator. Suitable systems may also be used toamplify the genetic material, but it is generally convenient to use E.coli for this purpose when only proliferation of the DNA is required.

Standard detection techniques well known in the art for detecting RNA,DNA, proteins and peptides can readily be applied to detect thymosin β15or its transcript to diagnose cancer, especially metastatic cancer or toconfirm that a primary tumor has, or has not, reached a particularmetastatic phase.

In one such technique, immunohistochemistry, anti-thymosin β315antibodies may be used to detect thymosin β15 in a biopsy sample.

Anti-thymosin β15 antibodies may also be used for imaging purposes, forexample, to detect tumor metastasis. Suitable labels includeradioisotopes, iodine (¹²⁵I, ¹²¹I) carbon (¹⁴C), sulphur (³⁵S), tritium(³H), indium (¹¹²In), and technetium (^(99m)Tc), fluorescent labels,such as fluorescein and rhodamine, and biotin.

However, for in vivo imaging purposes, the position becomes morerestrictive, as antibodies are not detectable, as such, from outside thebody, and so must be labelled, or otherwise modified, to permitdetection. Markers for this purpose may be any that do not substantiallyinterfere with the antibody binding, but which allow external detection.Suitable markers may include those that may be detected byX-radiography, NMR or MIR. For X-radiographic techniques, suitablemarkers include any radioisotope that emits detectable radiation butthat is not overtly harmful to the patient, such as barium or caesium,for example. Suitable markers for NMR and MIR generally include thosewith a detectable characteristic spin, such as deuterium, which may beincorporated into the antibody by suitable labelling of nutrients forthe relevant hybridoma, for example.

In the case of in vivo imaging methods, an antibody or antibody fragmentwhich has been labelled with an appropriate detectable imaging moiety,such as a radioisotope (for example, ¹³¹I, ¹¹²In, ^(99m)Tc), aradio-opaque substance, or a material detectable by nuclear magneticresonance, is introduced (for example, parenterally, subcutaneously orintraperitoneally) into the subject (such as a human) to be examined.The size of the subject, and the imaging system used, will determine thequantity of imaging moiety needed to produce diagnostic images. In thecase of a radioisotope moiety, for a human subject, the quantity ofradioactivity injected will normally range from about 5 to 20millicuries of technetium-99m. The labelled antibody or antibodyfragment will then preferentially accumulate at the location of cellswhich contain thymosin β15. The labelled antibody or antibody fragmentcan then be detected using known techniques.

The antibodies may be raised against either a peptide of thymosin β15 orthe whole molecule. Such a peptide may be presented together with acarrier protein, such as an KLH, to an animal system or, if it is longenough, say 25 amino acid residues, without-a carrier. Preferredpeptides include regions unique to thymosin β15, such as amino acid 7 to12 of SEQ ID NO: 2, amino acid 21 to 24 of SEQ ID NO: 2 and amino acid36 to 45 of SEQ ID NO: 2.

Polyclonal antibodies generated by the above technique may be useddirect, or suitable antibody producing cells may be isolated from theanimal and used to form a hybridoma by known means (Kohler and Milstein,Nature 256:795. (1975)). Selection of an appropriate hybridoma will alsobe apparent to those skilled in the art, and the resulting antibody maybe used in a suitable assay to identify thymosin β15.

Antibodies, or their equivalents, may also be used in accordance withthe present invention for the treatment or prophylaxis of cancers.Administration of a suitable dose of the antibody may serve to blockproduction, or to block the effective activity of thymosin β15, and thismay provide a crucial time window in which to treat the malignantgrowth.

Prophylaxis may be appropriate even at very early stages of the disease,as it is not known what actually leads to metastasis in any given case.Thus, administration of the antibodies, their equivalents, or factorswhich interfere with thymosin β15 activity, may be effected as soon ascancer is diagnosed, and treatment continued for as long as isnecessary, preferably until the threat of the disease has been removed.Such treatment may also be used prophylactically in individuals at highrisk for development of certain cancers, e.g., prostate.

A method of treatment involves attachment of a suitable toxin to theantibodies which then target the area of the tumor. Such toxins are wellknown in the art, and may comprise toxic radioisotopes, heavy metals,enzymes and complement activators, as well as such natural toxins asricin which are capable of acting at the level of only one or twomolecules per cell. It may also be possible to use such a technique todeliver localized doses of suitable physiologically active compounds,which may be used, for example, to treat cancers.

It will be appreciated that antibodies for use in accordance with thepresent invention, whether for diagnostic or therapeutic applications,may be monoclonal or polyclonal as appropriate. Antibody equivalents ofthese may comprise: the Fab′ fragments of the antibodies, such as Fab,Fab′, F(ab′)2 and Fv; idiotopes; or the results of allotope grafting(where the recognition region of an animal antibody is grafted into theappropriate region of a human antibody to avoid an immune response inthe patient), for example. Single chain antibodies may also be used.Other suitable modifications and/or agents will be apparent to thoseskilled in the art.

Chimeric and humanized antibodies are also within the scope of theinvention. It is expected that chimeric and humanized antibodies wouldbe less immunogenic in a human subject than the correspondingnon-chimeric antibody. A variety of approaches for making chimericantibodies, comprising for example a non-human variable region and ahuman constant region, have been described. See, for example, Morrisonet al., Proc. Natl. Acad. Sci. U.S.A. 81,6851 (1985); Takeda et al.,Nature 314,452(1985), Cabilly et al., U.S. Pat. No. 4,816,567; Boss etal., U.S. Pat. No. 4,816,397; Tanaguchi et al., European PatentPublication EP 171496; European Patent Publication 0173494, UnitedKingdom Patent GB 2177096B. Additonally, a chimeric antibody can befurther “humanized” such that parts of the variable regions, especiallythe conserved framework regions of the antigen-binding domain, are ofhuman origin and only the hypervariable regions are of non-human origin.Such altered immunoglobulin molecules may be made by any of severaltechniques known in the art, (e.g., Teng et al., Proc. Natl. Acad Sci.U.S.A., 80, 7308-7312 (1983); Kozbor et al., Immunology Today, 4, 7279(1983); Olsson et al., Meth. Enzymol., 92, 3-16 (1982)), and arepreferably made according to the teachings of PCT Publication WO92/06193or EP 0239400. Humanized antibodies can be commercially produced by, forexample, Scotgen Limited, 2 Holly Road, Twickenham, Middlesex, GreatBritain.

Another method of generating specific antibodies, or antibody fragments,reactive against thymosin β15 is to screen phage expression librariesencoding immunoglobulin genes, or portions thereof, with a protein ofthe invention, or peptide fragment thereof. For example, complete Fabfragments, V H regions and V-region derivatives can be expressed inbacteria using phage expression libraries. See for example Ward, et al.,Nature 341,544-546: (1989); Huse, et al., Science 246, 1275-1281 (1989);and McCafferty, et al., Nature 348, 552-554 (1990).

The antibody can be administered by a number of methods. One preferredmethod is set forth by Marasco and Haseltine in PCT WO94/02610, which isincorporated herein by reference. This method discloses theintracellular delivery of a gene encoding the antibody, in this case thethymosin β15 antibody. One would preferably use a gene encoding a singlechain thymosin β15 antibody, The antibody would preferably contain anuclear localization sequence, for example Pro-Lys-Lys-Lys-Arg-Lys-Val(SEQ ID NO:4) [Lawford, et al. Cell 46:575 (1986)];Pro-Glu-Lys-Lys-lle-Lys-Ser (SEQ ID NO:5) [Stanton, et al., Proc. Natl.Acad. Sci. USA 83:1772 (1986)], Gin-Pro-Lys-Lys-Pro (SEQ ID NO:6)[Harlow, et al., Mol. Cell. Biol. 5:1605 (1985)];Arg-Lys-Lys-Arg (SEQ IDNO:7) for the nucleus. One preferably uses an SV40 nuclear localizationsignal. By this method one can intracellularly express a thymosin β15antibody, which can block thymosin β15 functioning in desired cells.

In addition to using antibodies to inhibit thymosin β15, it may also bepossible to use other forms of inhibitors. Inhibitors of thymosin β15may manufactured, and these will generally correspond to the area of thesubstrate affected by the enzymatic activity. It is generally preferredthat such inhibitors correspond to a frozen intermediate between thesubstrate and the cleavage products, but it is also possible to providea sterically hindered version of the binding site, or a version of thebinding site which will, itself, irreversibly bind to thymosin β15.Other suitable inhibitors will be apparent to the skilled person.

The invention also provides for the treatment of a cancer by alteringthe expression of the thymosin β15. This may be effected by interferingwith thymosin β15 production, such as by directing specific antibodiesagainst the protein, which antibodies may be further modified to achievethe desired result. It may also be possible to block the thymosin β15receptor, something which may be more easily achieved by localization ofthe necessary binding agent, which may be an antibody or syntheticpeptide, for example.

Affecting thymosin β15 gene expression may also be achieved moredirectly, such as by blocking of a site, such as the promoter, on thegenomic DNA.

Where the present invention provides for the administration of, forexample, antibodies to a patient, then this may be by any suitableroute. If the tumor is still thought to be, or diagnosed as, localized,then an appropriate method of administration may be by injection directto the site. Administration may also be by injection, includingsubcutaneous, intramuscular, intravenous and intradermal injections.

Formulations may be any that are appropriate to the route ofadministration, and will be apparent to those skilled in the art. Theformulations may contain a suitable carrier, such as saline, and mayalso comprise bulking agents, other medicinal preparations, adjuvantsand any other suitable pharmaceutical ingredients. Catheters are anotherpreferred mode of administration.

Thymosin, β15 expression may also be inhibited in vivo by the use ofantisense technology. Antisense technology can be used to control geneexpression through triple-helix formation or antisense DNA or RNA, bothof which methods are based on binding of a polynucleotide to DNA or RNA.An antisense nucleic acid molecule which is complementary to a nucleicacid molecule encoding thymosin β15 can be designed based upon theisolated nucleic acid molecules encoding thymosin β15 provided by theinvention. An antisense nucleic acid molecule can comprise a nucleotidesequence which is complementary to a coding strand of a nucleic acid,e.g. complementary to an mRNA sequence, constructed according to therules of Watson and Crick base pairing, and can hydrogen bond to thecoding strand of the nucleic acid. The antisense sequence complementaryto a sequence of an mRNA can be complementary to a sequence in thecoding region of the mRNA or can be complementary to a 5′ or 3′untranslated region of the mRNA. Furthermore, an antisense nucleic acidcan be complementary in sequence to a regulatory region of the geneencoding the mRNA, for instance a transcription initiation sequence orregulatory element. Preferably, an antisense nucleic acid complementaryto a region preceding or spanning the initiation codon or in the 3′untranslated region of an mRNA is used. An antisense nucleic acid can bedesigned based upon the nucleotide sequence shown in SEQ ID NO: 1. Anucleic acid is designed which has a sequence complementary to asequence of the coding or untranslated region of the shown nucleic acid.Alternatively, an antisense nucleic acid can be designed based uponsequences of a β15 gene, which can be identified by screening a genomicDNA library with an isolated nucleic acid of the invention. For example,the sequence of an important regulatory element can be determined bystandard techniques and a sequence which is antisense to the regulatoryelement can be designed.

The antisense nucleic acids and oligonucleotides of the invention can beconstructed using chemical synthesis and enzymatic ligation reactionsusing procedures known in the art. The antisense nucleic acid oroligonucleotide can be chemically synthesized using naturally occurringnucleotides or variously modified nucleotides designed to increase thebiological stability of the molecules or to increase the physicalstability of the duplex formed between the antisense and sense nucleicacids e.g. phosphorothioate derivatives and acridine substitutednucleotides can be used. Alternatively, the antisense nucleic acids andoligonucleotides can be produced biologically using an expression vectorinto which a nucleic acid has been subcloned in an antisense orientation(i.e. nucleic acid transcribed from the inserted nucleic acid will be ofan antisense orientation to a target nucleic acid of interest). Theantisense expression vector is introduced into cells in the form of arecombinant plasmid, phagemid or attenuated virus in which antisensenucleic acids are produced under the control of a high efficiencyregulatory region, the activity of which can be determined by the celltype into which the vector is introduced. For a discussion of theregulation of gene expression using antisense genes see Weintraub, H. etal., Antisense RNA as a molecular tool for genetic analysis,Reviews—Trends in Genetics, Vol. 1 (1)1986.

In addition, ribozymes can be used to inhibit in vitro expression ofthymosin β15. For example, the nucleic acids of the invention canfurther be used to design ribozymes which are capable of cleaving asingle-stranded nucleic acid encoding a β15 protein, such as a thymosinβ15 mRNA transcript. A catalytic RNA (ribozyme) having ribonucleaseactivity can be designed which has specificity for an mRNA encodingthymosin β15 based upon the sequence of a nucleic acid of the invention(e.g., SEQ ID NO: 1). For example, a derivative of a Tetrahymena L-19IVS RNA can be constructed in which the base sequence of the active siteis complementary to the base sequence to be cleaved in a thymosinβ15-encoding mRNA. See for example Cech, et al., U.S. Pat. No.4,987,071; Cech, et al., U.S. Pat. No. 5,116,742. Alternatively, anucleic acid of the invention could be used to select a catalytic RNAhaving a specific ribonuclease activity from a pool of RNA molecules.See for example Bartel, D. and Szostak, J. W. Science 261,1411-1418(1993).

Methods for the diagnosis and prognosis of cancer using thepolynucleotides and antibodies of the present invention are set forth incopending application (Docket No. 46403) Express Mail No. TB338582354US,the disclosure of which is herein incorporated by reference.

All references cited above or below are herein incorporated byreference.

The following Examples serve to illustrate the present invention, andare not intended to limit the invention in any manner.

EXAMPLES Methods

Cell Culture

The poorly metastatic AT2.1 subline and high metastatic AT3.1, AT6.1 andMat Iylu sublines derived from Dunning R3327 rat prostaticadenocarcinoma cells (provided by Dr. J. Issaacs, The Johns HopkinsUniversity) were maintained in vitro in RPMI 1640 medium, supplementedwith 10% fetal bovine serum (Hyclone Laboratories, Logan, Utha), 1%glutamine/penicillin/streptomycin (Irvine Scientific, Santa Ana,Calif.), and 250 nM dexamethasome (Sigma Chemical Co, St. Louis, Md.),under an atmosphere of 5% CO₂; 95% air at 37° C.

RNA Isolation and Northern Blot Analysis

Cells at 70% confluency were harvested and subjected to RNA isolation.Total RNA was prepared by acid guanidinium thiocyanate/phenol/chloroformextraction procedures. (Chomczynski, P. & Sacchi, Anal. Biochem. 167,157-159 (1987)). Poly (A) RNAs were isolated from total RNA using Poly(A) Quik mRNA Isolation Kit (Stratagene, La Jolla, Calif.) or Micro FastTrack mRNA Isolation Kit (Invitrogen, San Diego, Calif.). 20 μg of totalRNA or 2 μg of mRNA was size fractionated on a denaturing formaldehydeagarose gel (1.1%) and transferred onto Hybond-N membrane (AmershamCorporation, Arlington Heights, Ill.) by capillary blotting in 0.05 MNaOH buffer according to the manufacturer's procedure. Northern blotfilters were prehybridized for 3 hours at 42° C. in 5×Denhardt's, 50%formamide, 5×SSPE, 0.5% SDS solution containing 100 μg/ml denaturedsalmon sperm DNA (Stratagene), followed by overnight hybridization infresh prehybridization solution with the addition of denatured probelabeled with [alpha-³²P] dCTP (New England Nuclear, Wilmington, Del.)using random primed DNA labeling kit (Boehringer Mannheim Biochemica,Indianapolis, Ind.). Filters were washed at increasing stringency to afinal stringency of 0.2×SSC; 0.1% SDS at 55° C. Autoradiography wasperformed over two days at −80° C. using Kodak X-Omat's film withintensifying screen. For reprobing, the original probe was removed bythe blots with boiling in 0.5% SDS water for 10 min.

mRNA Differential Display

DNase I digested 2 μg of total RNA from AT2.1, AT3.1 and AT6.1 cellsgrown to 70% confluency in RPMI 1640 medium supplemented with 10% fetalbovine serum and 250 nM dexamethasone were reverse-transcribed with 300units of MMLV reverse transcriptase (Stratagene) in the presence of 2.5μM of T 11 AG as primer and 20 μM dNTP for 60 min at 35° C. After heatinactivation of the reverse transcriptase at 95° C. for 5 min, 2 μl ofthe sample was amplified by PCR with T11 AG primer and arbitrary 10 mersin the presence of [α−³⁵S]dATP (New England Nuclear). The PCR parameterswere 94° C. for 30 sec, 42° C. for 1 min, and 72° C. for 30 sec for 40cycles, followed by 5 min elongation at 72° C. PCR products werefractionated on a 6% polyacrylamide gel and visualized byautoradiography. Differentially expressed bands were cut out of thedried gels and reamplified by PCR using the corresponding sets ofprimers. The reamplified PCR fragments were used as probes for Northernblot analysis.

cDNA Library Screening

An oligo(dT)-primed cDNA library was constructed in the lambda gt10vector (Amersham) using polyadenylated [poly(A)⁺] RNA obtained fromAT3.1 cells in culture. The library was screened with a ³²P-labeledprobe generated by PCR, using a 343 base pair AT3.1 cDNA isolated fromdifferential display as template. Filters were hybridized with probeovernight at 65° C. in a 5×Denhardt's, 5×SSPE, 0.5% SDS solutioncontaining 100 μg/ml denatured salmon sperm DNA, and washed at highstringency with 0.2× saline sodium citrate (SSC) and 0.1% SDS at 65° C.Inserts of positive clones were excised from λgt10 vector with EcoRIenzyme, subcloned into pbluescript II SK+/− (Stratagene) and sequencedusing the Sequenase Version 2.0 sequencing kit (U.S. Biochemical,Cleveland, Ohio).

RT-PCR Analysis

Total RNA from each cell line was digested with RNase free DNase I(GIBCO BRL, Gaithersburg, Md.). DNase I digested 5 μg of total RNA wasreverse transcribed using cDNA Cyling Kit (Invitrogen). The reversetranscrition mixture was purified with a Spin Column 300 (Pharmocia,Piscataway, N.J.). 10 μl of purified cDNA was amplified with primer setsof Tβ15 forward primer:

5′-TATCAGCTAGTGGCTGCACCCGCG-3′ (SEQ ID NO:8) and reverse primer:5′-AAATGCTGACCTTTCAGTCAGGGT-3′ (SEQ ID NO:9); Tβ4 forward primer:5′-ACTCTCAATTCCACCA TCTCCCAC-3′ (SEQ ID NO:10), reverse primer:5′-GCCTCTGAGCAGATCGTCTCTCCTTG-3′ (SEQ ID NO:11); and Tβ10 forwardprimer:

5′-ATAATATCCCTGGGCAAACCGGTG-3′ (SEQ ID NO:12), reverse primer:5′-GAGTGGAG TACCTGGAGCGCGAGC-3′ (SEQ ID NO:13), respectively. PCRamplification was performed in 50 μl of PCR reaction buffer (50 mM KCl,10 mM Tris [pH 8.5], 1.5 mM MgCl₂) with 1 mM of dNTPs, 50 pmol of eachprimer, and 2.5 U of Taq polymerase (GIBCO BRL), overlaid with 50 μl ofmineral oil (Sigma). The PCR profile was 94° C., 30 sec; 60° C., 30 sec;and 72° C., 2 min for 30 cycles. Control studies of the RT-PCR wereconducted using aliquats from the same samples and amplified withprimers to the β-actin gene (Clontech, Palo Alto, Calif.). Amplificationproducts were separated on 1.4% agarose gels.

In Situ Hybridization

Antisense and sense Tβ15 mRNA probes were prepared using Tβ15 cDNAinserted into the eukaryotic expression vector pcDNA3 (Invitrogen) astemplate and a digoxigenin RNA labeling kit (Boehringer Mannheim).Formalin-fixed paraffin-embedded sections were dewaxed, rehydrated, anddigested with proteinase K (50 μg/ml) in 100 mM Tris, 50 mM EDTA buffer(pH 8) for 8 min at 37° C. Hybridization was performed in an automatedinstrument (Ventana Medical Systems, Tuscon, Ariz.) for 60 min at 42° C.with 10 pM digoxigenin-labeled riboprobe in 100 μl of hybridizationbuffer (50% deionized formamide, 4×SSC, 10% dextran sulfate, 1% SDS, anddenatured herring sperm DNA (400 μg/ml)) per section under a liquidcover slip. The highest stringency of posthybridization washes was at45° C. for 15 min in 0.1×SSC. Bound digoxigenin-labeled probe wasdetected by anti-digoxigenin alkaline phosphatase conjugate andvisualized by nitroblue tetrazolium and5-bromo-4-chloro-3-indolylphosphate (NBT-BCIP) color reaction. Sectionswere counterstained with nuclear fast red.

GST-Two Fusion Protein Expression

PCR generated DNA fragments containing the full coding regions of Tβ15and Tβ4 were ligated in frame into the BamHI-EcoRI site of theprokaryotic expression vector pGEX-2T (Pharmacia, Piscataway, N.J.). ThepGEX-Tβ fusions were expressed in Escherichia coli, strain DH5α, byincubating with 0.1 mM isopropylthio-β-D-galactoside for 3 hours. Cellswere recovered by centrifugation, washed, and suspended in phosphatebuffered saline (PBS) containing 0.15 μ/ml aprotinin and 1mM EDTA andlysed by sonication. After addition of Triton X-100 to a finalconcentration of 0.1% (v/v), intact cells and debris were removed bycentrifugation. The supernatant was incubated with a 50% (v/v) slurry ofglutathione-agarose (Pharmacia) in PBS. After the beads were washed withexcess PBS and poured into a column, fusion proteins were eluted with asolution containing 50 mM Tris-HCI (pH 8.0) and 10 mM reducedglutathione (Sigma).

Actin Binding Experiment

Pyrene-labeled G-actin was prepared as previously described (Kouyama, etal., Eur. J. Biochem 114, 33-38 (1981). The final extents ofpolymerization were determined from the final levels of fluorescence ofpyrene-labeled antin as previously described (Janmey, et al.Biochemistry 24, 3714-3723 (1985).

Transfection

Tβ15 cDNA was cloned into pcDNA3 in either the sense or antisenseorientation relative to the constitutive human cytomegalovirus promoterand transfected into cells using lipofectin (GEBCO BRL, Gaithersburg,Md.). Individual stable transfectants were selected in media containing600 μg/ml of G418 (GIBCO BRL). Control transfections were done withpcDNA3 DNA devoid of Tβ15.

Cell Motility

Migration of transfectants was studied using a multiwell chamber assayas previously described (Kunda, et al., J. Cell Biol. 130, 725 (1995))48-well chemotaxis chambers were overlaid with 8-μm porositypolycarbonate filters (Nucleopore Corp., Pleasanton, Calif.) precoatedwith PBS containing 11.5 μg/ml fibronectin (Capple Organon Technica,Durham, N.C.). The migration of 5,000 cells placed in the upper welltoward fetal bovine serum in the lower well was assayed following a 4hour incubation at 37° C. After removal of cells from the upper side ofthe filters, cells that had passed through the filters and adhered tothe lower side were fixed in formalin, washed with PBS and stained withGill's triple strength hematoxylin (Polysciences, Warrington, Pa.) andcounted under light microscopy.

Generation of Polyclonal Antibody

0.25 mg of a synthetic oligopeptide (IQQEKEYNQRS) representing the 11carboxyl terminal amino acids of thymosin β15 dissolved in 380 ml of a0.125 M phosphate buffer, pH 7.4 was pipetted into reaction vesselcontaining 1.0 mg of keyhole limpet hemocyanin (Sigma). Then, 20 μl of25% aqueous glutaraldehyde solution was added. After gentle agitation,first for 3 h at room temperature and then for 12 h at 4° C., thereaction mixture was diluted with 0.15 M NaCI to a final concentrationof 100 μg/ml. The diluted mixture was then used for immunization. NewZealand White rabbits were immunized with 30 μg of the C-terminalpeptide of thymosin β15 as KLH conjugate emulsified with CFA. The firstbooster injection was given 6 weeks after the first immunization.Whereas subsequent booster injections were given at 3 weeks intervals.Production bleeds were obtained 2 weeks after the fifth boost. Antiserawere affinity purified over the C-terminal peptide conjugatedCNBr-activated Sepharose 4B column (Pharmacia) in 10 mM Tris-HCl, pH7.4. After extensive washing of the column with 0.5 M NaCl, 10 mM Tris,pH 7.4, the column was eluted with 0.2 M Glycine, 0.2 NaCl, pH 2.0. Thepurity and specificity of eluted fractions were examined by Westernanalysis.

Western Analysis

GST-Tβ fusion proteins were run on a 12% SDS-polyacrylamide gel andtransfered to a nitrocellulose membrane (0.2mm, Schleicher & Schuell,Keene, N.H.). The blot was incubated with 5% nonfat dry milk inphosphate-buffered saline containing 0.1% Tween 20 (TBS-T) followed byincubation with the 1:1000 diluted affinity purified anti Tβ15C-terminal peptide antidody for 1 h and washed 3 times with TBS-T. Theblot was then incubated with horseradish peroxidase-conjugatedanti-rabbit IgG antibody (Amersham Corp.) for 40 min, and a specificantibody reaction was detected by an enhanced chemiluminescencedetection system (Amersham Corp.).

Immunohistochemical Staining

Human prostate cancer sections were studied using an immunoperoxidaseABC kit (Vector, Burlingame, Calif.). Briefly, the 5 μm tissue sectionswere deparaffinized in xylene, rehydrated in graded alcohols, andblocked for endogenous peroxidase by 3% hydrogen peroxide (Sigma) inmethanol for 30 min. The sections were treated with normal goat serumfor 30 min and then incubated with an affinity purified anti Tβ15C-terminal peptide antibody for 2 h at room temperature at 1:100 (v/v)dilution, followed by incubation with a biotinylated goat anti-rabbitIgG antibody for 30 min. After incubation with a preformed ABC complexfor 30 min, specifically bound antibodies were visualized by usingperoxidase substrate, 3, 3′-diaminobenzidine tetrahydrochloride (DAB).Sections were counterstained with Gill's hematoxylin.

Results

Cloning of Tβ15

We compared patterns of gene expression by mRNA differential displayanalysis (Liang, P. & Pardee, A.B., Science 257, 967-971 (1992) in threevariants of the Dunning rat tumor: the weakly metastatic, poorly motileline AT2.1 and the highly metastatic, highly motile lines AT3.1 andAT6.1. One band, which was detected in the more motile AT3.1 and AT6.1lines by differential display (FIG. 1A) was confirmed by Northern (RNA)analysis to represent an overexpressed mRNA of approximately 420nucleotides in AT3.1, AT6.1 as well as the related MatLyLu cell line butwas not expressed in the poorly motile AT2.1 line (FIG. 1B). The genewas not expressed in other rat prostatic cell lines (non-metastatic)characterized by Northern analysis (data not shown).

To obtain a full-length complementary DNA (cDNA) clone of this gene, anAT3.1 cDNA library was screened using the originally cloned cDNAfragment from differential display as a probe. A positive clone with a412 base pair insert was isolated, which contained a single open-reading frame encoding a 45 amino acid protein with a calculatedmolecular mass of 5304 (FIG. 2). The insert size-of the clone wasapproximately the same as the molecular size of the transcript seen inNorthern analysis suggesting that the clone contained the full lengthgene sequence. A computer assisted homology search against the Genebankand EMBL DNA databases revealed that the novel gene shared 49%nucleotide sequence homology with rat thymosins β4 and β10. Alignment ofthe deduced amino acid sequence of the cloned gene with members of thethymosin β family (Mihelic, M. & Voelter, Amino Acids 6, 1-13 (1994)showed 68% homology with thymosin β4, 62% with thymosin β10 and 60% withβ9, β11 and β12 (FIG. 3). The results suggest that we have cloned anovel β thymosin, now named thymosin β15, from rat prostatic carcinomacells.

Hydropathy analysis of the thymosin β15 protein sequence revealed noapparent membrane-spanning or membrane-associated regions and noamino-terminal signal sequence. The protein is highly hydrophilic withan estimated isoelectric point of 5.14 and contains regions common toall members of the thymosin β family. All β-thymosin family memberspreviously studied, for example, have a putative actin binding region(LKKTET) 16 residues from the amino terminus (Vancompernolle, et al.,EMBO J. 11, 4739-4746 (1992), Troys, et al., EMBO J. 15, 201-210(1996).Thymosin 15 also has such a region, although the glutamic acid residueis replaced by an asparagine residue to form LKKTNT (FIG. 3). Theprincipal region of nonconformity between members of the thymosin βfamily occurs at the carboxyl terminus and the thymosin β15 sequence aswell shows no significant homology in this region with other familymembers.

Members of the β-thymosin family may be independently expressed indifferent tissues (Lin, et al., J. BioL Chem. 266, 23347-23353 (1991),Voisin, et al. J. Neurochem. 64, 109-120 (1995). Although thymosin β15is differentially expressed in the prostate carcinoma cell lines tested,all of these lines expressed equivalent levels of thymosins β4 and β10by RT-PCR analysis (FIG. 11). The tissue distribution of thymosin β15mRNA was examined in the major organs of the rat. No expression ofthymosin β15 was detected in the heart, brain, lung, spleen, liver,skeletal muscle and kidney, whereas high expression was found in thetestis (FIG. 4). Southern (DNA) analysis of Hind III-, EcoR I- and PstI-restricted DNA from AT2.1 and AT3.1 cells with thymosin β15 cDNA proberevealed no gross structural alteration of the thymosin β15 gene in thetumor cells (data not shown). These results demonstrate that a novelmember of the thymosin β family is upregulated in metastatic ratprostatic carcinoma cell lines, whereas expression of other thymosin βfamily members (β4 and β10) remains unchanged.

Cloning of Human Thymosin β15 by RT-PCR

DNase I digested 5 μg of total RNA from human prostatic carcinoma cellline PC-3 was reverse transcribed using cDNA Cycling Kit (Invitrogen).The reverse transcription mixture was purified with a Spin Column 300(Pharmocia, Piscataway, N.Y.). 10 μl of purified cDNA reaction wasamplified with primers F1 (5′-TATCAGCTAGTGGCTGCACCCGCG-3′) (SEQ ID NO:8)and RI (5′-AAATGCT GACCTTTCAGTCAGGGT-3′) (SEQ ID NO:9) designed toanneal to the outer ends of the thymosin β15 sequence. PCR amplificationwas performed in 50 μl of PCR reaction buffer (50 mM KCl, 10 mM Tris [pH8.5], 1.5 mM MgCl2) with 1 mM of dNTPs, 50 pmol of each primer, and 2.5U of Taq polymerase (GIBCO BRL), overlaid with 50 μl of mineral oil(Sigma). The PCR profile was 94° C., 30 sec; 60° C., 30 sec; and 72° C.,2 min for 30 cycles. Control studies of the RT-PCR were conducted usingaliquats from the same samples and amplified with primers to the β-actingene (Clontech, Palo Alto, Calif.). Amplification products wereseparated on 1.6% agarose gels. The amplified PCR product was ligated topCR using TA cloning kit (Invitrogen, San Diego, (Calif.), and then DNAsequenced. The sequence of the PCR product of human prostatic carcinomacells amplified by the thymosin β15 primers was surprisingly 100%identical to the thymosin β15 sequence obtained from the rat prostaticcarcinoma cells.

Expression of Tβ15 mRNA in Human Prostate Cancer

To determine whether this thymosin family member may be expressed inhuman prostate cancer, we examined human prostatic carcinoma cell linePC-3 by RT-PCR with forward and reverse primers for thymosin β15. ThePC-3 cells showed a low level of thymosin β15 expression. The DNAsequence of the amplified PCR product was 100% identical to the ratthymosin β15 sequence. We conducted in situ hybridization study onsamples from patients with varying grades of prostatic carcinomas usinga thymosin β15 probe. The tissue sections allowed direct comparison ofnormal and malignant elements on the same samples. The stromal elementswithin and around the tumor cell masses, as well as the nonmalignantprostatic epithelium adjacent to the tumor showed little backgroundhybridization with the thymosin β15 antisense probe. In contrast,specific tumor cell islands exhibited a strong specific thymosin β15signal when probed with antisense (FIG. 5A, small arrow) but not with asense RNA probe (data not shown). Although nearly all of the tumor cellsin the positive islands expressed thymosin β15 mRNA, not all patientspecimens were positive and not all islands in a single prostate werepositive (FIG. 5A, large arrow). The majority of the negative tumorcells were in non-invasive in situ carcinomas whereas highly invasivetumors were consistently positive (FIG. 5B). Thus a novel β thymosin,first detected in metastatic rat prostate carcinoma cell lines, isupregulated in human prostate cancer.

Effect of Tβ15 on Actin Polymerization

Because thymosin β15 retains a putative actin-binding domain, we testedits effect on actin polymerization using recombinant fusion proteins.The results, shown in FIG. 6A, reveal that a glutathione-S-transferase(GST)/thymosin β15 fusion protein inhibits polymerization ofpyrene-derivatized actin monomers to an equal or slightly greater extentthan a GST/thymosin b4 fusion protein, suggesting that these twoproteins have similar actin-sequestering properties. Similar resultswere obtained when thymosin β15 was cleaved from the GST-fusion proteinwith thrombin and subsequently analyzed for its ability to inhibit therate and extent of actin polymerization (FIG. 6B and C). The differencein apparent affinity for actin between free and GST-fused thymosin β15is likely related to the GST-mediated dimerization of the fusionpeptides to form complexes with two actin monomer binding sites thateither bind actin more tightly or bind to the end of the growingfilament, thereby inhibiting polymerization at low molar ratios to totalactin. One example of such an effect is the strong retardation of actinassembly by actobindin, which appears to function as a dimer ofthymosin-like actin binding sites (Bubb, et al., Biochemistry 34,3921-3926 (1995).

Effect of Tβ15 on Cell Motility

To determine whether thymosin β15 expression had an effect on cellmotility, we transfected highly motile AT3.1 cells with a eukaryoticexpression vector (pcDNA3) containing the thymosin β15 gene in antisenseorientation driven by the constitutive human cytomegalovirus promoter.The transfected cells growing in selective (G418) media were examinedfor expression of antisense transcripts of the thymosin β15 gene bystrand-specific polymerase chain reaction (PCR) amplification (Zhou, etal., Cancer Res. 52, 4280-4285 (1992). Analysis of cell motility in amultiwell Boyden chamber apparatus (Boyden, S. V., J. Exp. Med. 115,453-466 (1962)) using fetal bovine serum as a migration stimulusrevealed that the motility of the transfectants which showed expressionof antisense transcripts was significantly reduced relative to thevector-only controls (FIG. 7A). Two antisense transfected clones whichdid not express antisense transcripts failed to show any decreased rateof cell motility (data not shown). In a further experiment, poorlymotile AT2.1 cells, transfected with sense thymosin β15 constructs andconfirmed to express thymosin β15 by Northern analysis, were shown tohave significantly increased stimulated motility relative to theirvector controls (FIG. 7B). Both the sense and antisense thymosin β15transfectants showed similar rates of cell proliferation relative tocontrols suggesting differential specificity for different cellularevents (FIG. 7C). The results demonstrate that thymosin β15, which isupregulated in the highly motile AT3.1 and AT6.1 Dunning tumor celllines, is a positive regulator of cell motility which is an importantcomponent of cancer metastasis.

Immunohistochemical Detection of Tβ15 in Prostate Carcinoma

A polyclonal antibody was raised against a peptide representing the 11C-terminal amino acids of thymosin β15. Synthesized peptide was coupledwith a carrier, keyhole limpet hemocyanin (KLH), and injected intorabbits. Antiserum was affinity-purified over the C-terminal peptidecoupled CNBr-activated sepharose 4B column. To test the specificity ofthe purified antibody, we performed Western analysis of the GST/thymosinβ fusion proteins with the affinity-purified anti C-terminal antibody.The purified antibody strongly reacted with GST-thymosin β15 fusionprotein, but did not cross react with GST-thymosin β4, and not with GSTalone (FIG. 8) showing its specificity.

We used the affinity purified polyclonal thymosin β15 antibody forimmunohistochemical study of human prostate carcinoma. The results aresummarized below in Table 1. The thymosin β15 immunostaining wasobserved in the cytoplasms of epithelial cells in neoplastic prostatesbut not in normal prostates and not in the stromal cells (FIG. 10A,large arrow). Among the investigated malignant epithelia, the poorlydifferentiated prostate carcinomas displayed the most extensive andintense thymosin β15 immunoreaction (FIG. 10C), followed by moderatelydifferentiated prostate carcinomas in which not all carcinomas expressedthymosin β15 showing partial positivity (FIG. 10B). In some cases,high-grade prostatic intraepithelial neoplasia (PIN) showed thymosin β15immunostaining, but to a lesser extent FIG. 10A, small arrow). In poorlydifferentiated invasive carcinoma, single cells invading stromadisplayed intense staining (FIG. 10D). The expression of thymosin β15well correlated with Gleason grade of prostate carcinoma.

TABLE 1 THYMOSIN β15 EXPRESSION IN HUMAN PROSTATE CARCINOMA Prostate No.Negative^(a) Partial^(b) Positive^(c) BPH 2 2 0 0 Ca Gleason 2˜5 5 3 2 0Ca Gleason 6˜8 25 4 7 14 Ca Gleason 9˜10 6 0 1 5 Ca (with met) 3 0 1 2(BPH - Benign Prostate Hyperplasia; Ca - Carcinoma) ^(a)less than 10%cells showing positivity ^(b)heterogeneous staining with 30˜75% of cellsshowing positivity ^(c)homogeneous staining with 75˜100% of cellsshowing positivity

Expression of Thymosin β15 mRNA in Mouse Lung Carcinoma

To determine whether thymosin β15 may be expressed in other kind ofcancer cells, we tested mouse lung carcinoma cell lines by Northernanalysis. The results showed the thymosin β15 expression in metastaticcell lines M27 and H59, but showed no expression in a nonmetastatic cellline LA-4 (FIG. 9).

Discussion

Progression to the metastatic stage is directly correlated withmortality from prostatic carcinoma. It therefore follows that the earlydiagnosis, prevention, or therapeutic treatment of metastaticprogression would lead to more effective control of this disease. TheDunning R-3327 rat prostatic adenocarcinoma model provides severalsublines with varying metastatic ability, all of which derive from anoriginal spontaneous tumor and which provide an opportunity to study thesteps leading to prostate cancer metastases (Mohler, Cancer Metast. Rev.12, 53-67 1993) and Pienta, et al. Cancer Surveys 11, 255-263 (1993)).By comparing gene expression among the Dunning cells, we cloned a novelmember of the thymosin β family, thymosin β15, which is expressed inhighly metastatic prostate cancer cells but not in non- or weaklymetastatic cells. The related family members thymosin β4 and β10 areexpressed equally in all of the cell lines tested such that theirexpression does not vary with increasing metastatic potential.

Thymosin β15 binds G-actin and retards actin polymerization. Because thehighly motile prostate cancer cell lines showed high level expression ofthymosin β15, we tested whether thymosin β15 transfection into theDunning rat carcinoma cell lines could influence cell motility. Ourresults show clearly that transfection of sense or antisense thymosinβ15 constructs into rat prostatic carcinoma cells can significantlymodulate stimulated cell migration, a property not previously associatedwith β-thymosins. In cancer, the enhanced movement of malignant tumorcells through connective tissues is a major contributor to progressiontoward the metastatic stage. In order to metastasize, a tumor cell mustinitially dissociate from the primary tumor, migrate through connectivetissue and capillary walls into the circulatory system, and migrateagain across the vascular wall into a secondary site. Therefore,increases in thymosin β15 expression in malignant prostate carcinomacells are believed to mediate an important change in tumor progressiontoward metastasis and that the expression of thymosin β15 is a usefulmarker for diagnosis and prognosis of cancer malignancy.

Cell motility is typically associated with coordinated disassembly andreformation of the cortical actin network (Cunningham, et al., Science251, 1233-1236 (1991), Haugwitz, et al., Cell 79, 303-314 (1994) andStossel, Science 260, 1086-1094 (1993)). Enhanced expression oractivation of thymosin's actin binding function may therefore stimulatemotility by enhancing the depolymerization phase of this process. Thefinding that a molecule which acts to retard actin polymerization maystimulate cell motility is consistent with the recent finding of Hug etal. (Hug, et al., Cell 81, 591-600 (1995) which showed that overexpression of an action capping protein in Dictyostelium cells led to anincreased rate of cell motility. The findings on the relationshipbetween actin depolymenzation and increased motility also support ourhypothesis that the upregulation of thymosin β15 may represent animportant step in the progression of prostatic carcinoma to themetastatic state. The finding that thymosin β15, which is upregulated inmore highly metastatic rat prostate cancer cell lines, is alsoupregulated in human prostate cancer is intriguing. At present, the bestmarkers for prostate cancer, such as PSA expression, are most useful forearly detection of prostate cancer. However, they do not allow anydistinction of metastatic tumor from non-metastatic tumors.

This invention has been described in detail including the preferredembodiments thereof. However, it will be appreciated that those skilledin the art, upon consideration of this disclosure, may makemodifications and improvements thereon without departing from the spiritand scope of the invention as set forth in the claims.

13 412 base pairs nucleic acid single linear cDNA NO NO not providedCoding Sequence 98...232 Exon 1 1 TATCAGCTAG TGGCTGCACC CGCGAACACCACCCTGGTCC GGAGTAGCTG CGGACAGAAT 60 TGCTGGCCTA GTAGAAGCTT TGGAACGAGCAGTCAAG ATG AGT GAT AAA CCA GAC 115 Met Ser Asp Lys Pro Asp 1 5 TTA TCAGAA GTT GAA ACA TTT GAC AAA TCA AAG TTG AAG AAG ACT AAT 163 Leu Ser GluVal Glu Thr Phe Asp Lys Ser Lys Leu Lys Lys Thr Asn 10 15 20 ACT GAA GAAAAG AAT ACT CTT CCT TCG AAG GAA ACT ATC CAG CAG GAG 211 Thr Glu Glu LysAsn Thr Leu Pro Ser Lys Glu Thr Ile Gln Gln Glu 25 30 35 AAA GAA TAT AATCAA AGA TC ATAAAATGAG ATTCTCCTCT CAAGAGCAAC TTCAAC 267 Lys Glu Tyr AsnGln Arg Ser 40 45 TTTGCTGGAT AGTCTTGGAT TTAGACATGT TTCTGTAAAC CTATCCAATATGTAGACATT 327 TTAGGCGGTT CCTGATAGGT TCTTAAGTAC CCTGACTGAA AGGTCAGCATTTAACACCAA 387 TCATTAAATG TGTTTTCCAC TGCTC 412 45 amino acids amino acidsingle linear protein NO NO internal not provided 2 Met Ser Asp Lys ProAsp Leu Ser Glu Val Glu Thr Phe Asp Lys Ser 1 5 10 15 Lys Leu Lys LysThr Asn Thr Glu Glu Lys Asn Thr Leu Pro Ser Lys 20 25 30 Glu Thr Ile GlnGln Glu Lys Glu Tyr Asn Gln Arg Ser 35 40 45 10 base pairs nucleic acidsingle linear cDNA NO NO not provided 3 AGGGAACGAG 10 7 amino acidsamino acid single linear peptide NO NO N-terminal not provided 4 Pro LysLys Lys Arg Lys Val 1 5 7 amino acids amino acid single linear peptideNO NO N-terminal not provided 5 Pro Glu Lys Lys Ile Lys Ser 1 5 5 aminoacids amino acid single linear peptide NO NO N-terminal not provided 6Gln Pro Lys Lys Pro 1 5 4 amino acids amino acid single linear peptideNO NO N-terminal not provided 7 Arg Lys Lys Arg 1 24 base pairs nucleicacid single linear cDNA NO NO not provided 8 TATCAGCTAG TGGCTGCACC CGCG24 24 base pairs nucleic acid single linear cDNA NO NO not provided 9AAATGCTGAC CTTTCAGTCA GGGT 24 24 base pairs nucleic acid single linearcDNA NO NO not provided 10 ACTCTCAATT CCACCATCTC CCAC 24 26 base pairsnucleic acid single linear cDNA NO NO not provided 11 GCCTCTGAGCAGATCGTCTC TCCTTG 26 24 base pairs nucleic acid single linear cDNA NO NOnot provided 12 ATAATATCCC TGGGCAAACC GGTG 24 24 base pairs nucleic acidsingle linear cDNA NO NO not provided 13 GAGTGGAGTA CCTGGAGCGC GAGC 24

What is claimed is:
 1. An isolated antibody or antibody fragment which selectively binds to a thymosin β15 protein, said thymosin β15 protein having been obtained from a host cell containing a DNA segment having the nucleotide sequence set forth in SEQ ID NO:
 1. 2. The isolated antibody or antibody fragment of claim 1, wherein the DNA segment encodes a protein encoding the amino acid sequence of SEQ ID NO:
 2. 3. An isolated antibody or antibody fragment which selectively binds to a protein comprising an amino acid sequence selected from the group consisting of amino acid 7 to 12 of SEQ ID NO:2, amino acid 21 to 24 of SEQ ID NO:2 and amino acid 36 to 45 of SEQ ID NO:2.
 4. The antibody fragment of claims 1 or 3 wherein said fragment is a Fab, Fab′, F(ab′) or Fv fragment.
 5. The antibody of claims 1 or 3 wherein said antibody is a single chain antibody.
 6. The antibody of claims 1 or 3 wherein said antibody is humanized.
 7. The antibody or antibody fragment of claims 1 or 3 wherein said antibody or antibody fragment is detectably labelled. 